A large-scale analysis of genome sequences of various organisms such as human has been studied, and its goal is almost achieved. It is a next task to elucidate functions of proteins, which are encoded in enormous numbers of genes discovered by the sequence analysis. Findings obtained by these protein functions are expected to be greatly useful for the development of new drugs. The analysis of the three dimensional structure of protein provides useful information for elucidating the protein function or drug design. Hereafter, its importance will be increasing and a high throughput analysis in accordance with a large scale analysis will be desirable.
A purified protein in milligram order is necessary for the analysis of the three dimensional structure of protein. Previously, a large-scale preparation of protein was a bottle-neck for the three dimensional structural analysis, however, a desired protein can be easily prepared in large-scale, by the advanced gene cloning techniques using an expression system such as that of a microorganism or a cultured cell at the moment. Further, a cell-free protein synthesis system has been improved by various methods such as dialysis and the like, to obtain a protein in milligram order for several hours. Thus, the high throughput analysis of three dimensional structure of protein is coming true.
However, these methods are not always applicable to every kind of proteins, and it is still difficult to prepare a protein that has a hydrophobic region, such as a membrane protein, in large amount. In the expression system of cultured cell, a membrane protein is accumulated in the cell membrane by the localization system of the host cell. Thus, when the expressed protein is purified from the cultured cell, a step to extract the protein from the membrane by using detergents is necessary. This step needs much expense in time and effort, and is not so efficient in the extraction. Some kinds of detergent often impair the intrinsic structure and function of the protein. When a protein that has a hydrophobic region therein, such as a membrane protein is expressed in E. coli, the expressed protein often forms insoluble precipitation. Therefore, it is necessary to solubilize the precipitate by using a strong denaturating agent such as guanidine salts or urea, and to renaturate or refold the denatured protein to its intrinsic structure (a folding step) in the purification. These steps are also at much expense in time and effort, and have many problems such as the reprecipitation during the folding step. These proteins also form an insoluble precipitate in a cell-free protein synthesis system, so that the amount of the synthesized protein is not sufficient.
As described above, the problem to solubilize membrane proteins makes it difficult to prepare a large amount of the proteins, and retards the analysis of three dimensional structures of the proteins under the current circumstances. Among membrane proteins, however, there are many important proteins for the target of drug development such as a receptor, channel protein, transporter and the like. The structural analysis of these proteins is urgently necessary for the efficient development of the drug.